Male Sprague–Dawley rats each weighing 350-400 g were used (Charles River Labs, Raleigh, NC, United States). Animals were housed in a humidity and temperature controlled (22.2 ± 0.2°C) room with free access to food and water. All animals were handled as outlined in the Guide for the Care and Use of Laboratory Animals (ISBN: 0–309–05377-3) and animal protocol was approved by the National Institute of Drug Abuse Animal Care and Use Committee (NIDA, ACUC).

Rats were anesthetized using ketamine (100 mg/kg i.p.) and xylazine (5 mg/kg i.p.) and silastic catheters were put into the jugular vein. After surgery, animal health was monitored daily, and catheters were flushed with sterile saline containing gentamicin (Butler Schein; 5 mg/ml) and allowed to recover for 5–10 days before start of METH self-administration training. Upon wake from anesthesia, rats received meloxicam (1 mg/kg s.c.), as an analgesic, and a second dose the following day.

As mentioned, our METH self-administration training procedure was performed according to the previously designed protocol for our lab (Cadet et al., 2016; Subu et al., 2020; Jayanthi et al., 2022). Rats were housed in self-administration chambers with free access to food and water, made available in water bottles and feeders hanging from each chamber wall. We trained rats to self-administer dl-methamphetamine HCl (NIDA), by pressing an active infusion pump lever, for three 3-h sessions per day, for 21 days. To achieve this, rat catheters were connected to a modified cannula (Plastics One, Minneapolis, MN) attached to a liquid swivel (Instech Laboratories, Inc., Plymouth Meeting, PA, United States) via polyethylene-50 tubing protected by a metal spring. To prevent overdose, each 3-h self-administration training session was separated by a 30-min timeout during which the animals had no access to the active lever. Rats self-administered METH at a dose of 0.1 mg/kg/infusion over 3.5 s, and the number of infusions was limited to 35 per each 3-h training session, and each pressed lever infusion was given a 20-s recess timeout. To reinforce, active lever presses were accompanied by a compound tone-light cue, and presses on inactive lever induced no reinforcement cues. Rats were trained 5 days a week, with 2 days off to minimize weight loss for a total of 21 days of METH self-administration training. During the 2 days off, rats remained housed in chambers but were disconnected from the intravenous self-administration connection and cannulas were covered with dust prevention caps. We started self-administration sessions at the onset of the dark cycle and sessions began with insertion of the active lever and the illumination of a red light that remained on during the entire 3 h session. At the end of each session, and during 30-min timeouts, the red light was turned off, and the active lever was removed. Control rats self-administered saline, under the same conditions.

During the 8-day foot-shock punishment phase, rats continued METH self-administration for 8 days straight under the same 3-h session schedule used during the training phase. However, a 0.5-s foot-shock was delivered through the grid floor on 50% of all active METH lever presses. This electrical current was set at 0.18 mA on day 1, which was increased as the 8-day foot-shock phase proceeded. Shock intensity was set to 0.24 mA on day 2, 0.30 mA on days 3–5, and 0.36 mA on days 6–8. As an important addition, some control rats that self-administered saline were “yoked” connected to a METH taking rat and received a contingent shock whenever the rat it was paired to pressed for METH. As a control for the effects of shock on biochemical and molecular markers within the brain, the METH taking rat and the yoke-saline rat that it was paired to, received the same number of foot-shocks. Totally, after the 8-day foot-shock phase was complete, rats were separated into 5 groups CT (Saline, no foot-shock), SR (Methamphetamine, Shock Resistant), SS (Methamphetamine, Shock Sensitive), YSR (Saline, SR contingent foot-shock), and YSS (Saline, SS contingent foot-shock).

Immediately after the last 3 h foot-shock session, rats were euthanized by decapitation, and the brains were removed and dissected by region. We dissected out the entire hippocampus using the coordinates A/P -5 to -7 mm bregma, mediolateral ±6 mm, D/V -2 to -8 mm, corresponding to The Rat Brain in Stereotaxic Coordinates (Paxinos and Watson, 2013).Total RNA was extracted from samples of the total hippocampal region using Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, United States). RNA integrity was assessed using an Agilent 2,100 Bioanalyzer (Agilent, Palo Alto, CA, United States), and RNA samples showed no signs of degradation. RNA sequencing was performed by GeneWiz (GeneWiz, South Plainfield, NJ, United States) using Illumina HiSeq instrument according to manufacturer’s instructions and was converted into fastq files and de-multiplexed using the Illumina bcl2fastq 2.17 software. The samples were sequenced using a 2×150bp Paired End (PE) configuration (GeneWiz, South Plainfield, NJ, United States). Sequence reads were filtered to remove any poor-quality reads using Trimmomatic v.0.36 and this data has been submitted to Gene Expression Omnibus¹ under accession number GSE203268.

We used Ingenuity Pathway Analysis (IPA) software (Qiagen, Valencia, CA, United States) to analyze genes that showed differential expression in RNA sequencing data and identify molecular functions, biochemical networks, and validated canonical gene pathways.

To analyze mRNA levels, for each sample in each group (CT, SR, SS, YSR, YSS), we reversed-transcribed individual total RNA into cDNA using Advantage RT-for-PCR kit (Clontech, Mountain View, CA, United States). Using this kit, 500 nanograms(ng) of RNA was reversed-transcribed with oligo dT primers to create cDNA. We generated PCR primers using NIH accredited NCBI primer-BLAST website and ordered gene-specific primers from the Synthesis and Sequencing Facility of Johns Hopkins University (Baltimore, MD, United States). RT-qPCR was performed with the Roche LightCycler 480 II using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules CA, United States). Relative amounts of mRNA in each sample were normalized to a combined mean of OAZ1 and Clathrin mRNA, used as housekeeping reference genes.

Behavioral data, RNA sequencing data, and qRT-PCR data were analyzed using the statistical program GraphPad Prism version 9 (Dotmatics, San Diego, CA, United States). Behavioral data was analyzed using repeated measures two-way ANOVA. For the behavioral experiments, the dependent variable used was the number of METH or saline infusions during the 21-day training and 8-day foot-shock phases, for each group. RNA sequencing and qRT-PCR data was analyzed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test. Statistical significance level for all tests was set to 0.05, and the null hypothesis was rejected at value of p<0.05.

Figure 1A shows the experimental timeline of the behavioral experiment. It includes METH SA training and contingent foot-shock phases. Figure 1B depicts the number of infusions of either METH or saline during the 21-day SA training period. All METH-trained rats (n = 15) significantly increased their drug intake whereas control rats (n = 9) did not change their saline intake during the training phase (Figure 1B). The repeated measure analysis using 2-way ANOVA for the SA training phase included the between-subject factor of groups: control (CT n = 9), yoked saline (YS n = 19), shock-resistant (SR n = 8), and shock-sensitive (SS n = 7), and the within-subject factor of training days (experimental days 1–21). There were significant differences in the number of infusions between groups [F₍₃,₃₅₎ = 139.9, p < 0.0001] over training days [F₍₂₀,₇₀₀₎ = 34.29, p < 0.0001], with there being significant interaction of day × group [F₍₆₀,₇₀₀₎ = 21.31, p < 0.0001].

On day 22, foot-shocks were introduced and increased in intensity from 0.18–0.36 mA over 8 days (experimental days 22–29). Foot-shocks helped to separate the rats into 2 different behavioral phenotypes. The group of rats that continued to compulsively press the active lever for METH infusions despite foot-shocks were labeled shock-resistant (SR) or compulsive METH takers (n = 8) whereas the rats that decreased their active lever pressing under punishment were termed shock-sensitive (SS) or non-compulsives (n = 7; Figure 1B). The repeated measures analysis for the foot-shock phase included the SR and SS groups, and the within-subject factor of days (experimental days 22–29). We found significant differences in the number of METH infusions between groups [F_(1,13) = 46.53, p < 0.0001] and over shock days [F_(7,91) = 6.654, p < 0.0001]. The interaction of day × group [F_(7,91) = 9.348, p < 0.0001] was also significant.

The total METH intake (mg/kg) of the SR and SS rats during the training and foot-shock phases are shown in Figure 1C. During SA training, all METH SA rats took comparable amounts of the drug. During the foot-shock/punishment phase, both groups decreased their drug intake, with the SS rats decreasing their active lever pressing significantly more than the SR rats. Overall, SS rats took significantly less METH [F_(1,6) = 66.86; p = 0.0002; Figure 1C] and received significantly less foot-shocks [F_(1,13) = 64.69; p < 0.0001; Figure 1D] than SR rats. The 2-way ANOVA for number of foot-shocks was also significant for groups [F_(1,13) = 64.69, p < 0.0001], days [F_(7,91) = 6.920, p < 0.0001], and interaction of day × group [F_(7,91) = 9.892, p < 0.0001; Figure 1D].

As mentioned in the Methods, some rats that self-administered saline were individually connected to METH taking rats. These yoked rats received foot-shocks whenever the METH SA rat to which they were paired received a shock, such that individual yoked-saline rats and paired METH-taking rats received the same number of shocks by the end of the behavioral experiment. These paired rats were labeled yoked shock-resistant (YSR) or yoked shock-sensitive (YSS).

To identify potential transcriptional changes in the resistant and sensitive rats, we used RNA sequencing using RNA obtained from whole hippocampal tissues (CT, n = 7; SR, n = 7; SS, n = 6; YSR, n = 5; YSS, n = 5). We then calculated RNA expression fold changes to find differentially expressed genes (DEGs) between 8 pairings (SR vs. CT, SS vs. CT, YSR vs. CT, YSS vs. CT, SR vs. SS, SR vs. YSR, SS vs. YSS, YSR vs. YSS).

Using GraphPad Prism (Version 9), we created volcano plots to visualize the entire sets of DEGs obtained from the sequencing data (Figures 2A,H). The volcano plots, showing up- and downregulated genes, were plotted using fold-changes scaled on the x-axis, and value of p shown on the y-axis. Note that SR group comparisons (SR vs. CT and SR vs. YSR; Figures 2A,F) showed more total DEGs than the SS group comparisons (SS vs. CT and SS vs. YSS; Figures 2B,G). The YSR vs. CT (Figure 2C) and YSS vs. CT (Figure 2D) comparisons showed clearly that shock-induced stress had significant effects on hippocampal gene expression.

To identify potential overlaps of DEGs between the various comparisons, we created Venn diagrams² (Figures 2I,J). For these comparisons, we used fold-changes of greater or less that 1.7-fold and p-values of 0.05. We found that 30 upregulated genes (Figure 2I) and 9 downregulated genes (Figure 2J) were unique to the SR vs. SS comparison. Figures 2I,J showed very little overlaps between the pairwise comparisons.

We used DAVID gene functional annotation tool (DAVID Bioinformatics) to examine functional classifications for the significant DEGs among the eight comparisons (Figure 2K). The DEGs fall within functional classes that included cell signaling, metal-binding, transport of ions, calcium signaling, and cell adhesion (Figure 2K). Figure 2L shows a heatmap (GraphPad Prism) that illustrates relative changes in mRNA expression in the 8 pair-wise comparisons.

Kyoto encyclopedia of genes and genomes pathway analysis was also used to further identify relevant pathways that were differentially impacted in the compulsive and non-compulsive rats. Many DEGs participated in pathways related to neuronal ligand-receptor interactions, neuroinflammation, metabolism and absorption, and CAMs (Figure 3A).

We used Sankey diagrams to further show the interactions of up- (Figure 3B) and downregulated (Figure 3C) genes located in various KEGG pathways. The diagrams show that single genes that participate in multiple functions in the hippocampus are impacted by compulsive METH taking behaviors.

We also used Ingenuity Pathway Analysis (IPA) to identify biological networks affected by METH SA and foot-shocks (Figures 4A,B). Figure 4A showed upregulation of glycosylation dependent cell adhesion molecule (Glycam1) and myelin protein zero-like 2 (Mpzl2) in the comparison between the shock-resistant vs. -sensitive rats. Figure 4B showed that there was increased expression of L-dopachrome Tautomerase (Dct) in the resistant group in comparison to the sensitive rats.

To further validate the RNA sequencing results, we ran qRT-PCR using primers for some CAMs of interest. The PCR results showing fold-changes in mRNA levels are shown in Figures 4C–H. Cdh1 (cadherin 1), a member of the cadherin family of CAMs was significantly decreased [F_(4,26) = 3.445; p = 0.0219] in the SS group compared to SR (Figure 4C). Mpzl2 [F_(4,25) = 9.393; p < 0.0001], Glycam1 [F_(4,23) = 18.62; p < 0.0001], and Dct [F_(4,27) = 6.290; p < 0.0010], all showed significant increases in the compulsive group in comparison to the other groups (Figures 4E–G).